The aim of this project is to further elucidate the mechanisms of action of intracellular messengers such as calcium ion, cAMP and cGMP on electrical properties of nerve cell membranes, in particular the membrane conductances. Experiments will be done primarily on molluscan ganglion cells, utilizing voltage clamp in conjunction with injection and perfusion methods, and local current recording with a surface patch clamp pipette. In parallel with these approaches, a major effort will go into the development of a new means of rapidly and uniformly perturbing the intra-cellular concentration of Ca2ion. A similar method may also prove feasible for cAMP and cGMP. We will first seek a specific high-affinity Ca-complexing ligand with Ca-buffering properties similar to EGTA, but that undergoes an increase in dissociation when irradiated with strong light of appropriate wavelength. Such a light-modulated Ca buffer injected into the cell will allow us to change the intracellular pCa rapidly and uniformly with light pulses. In parallel we will work to develop a light-modulated cAMP agonist that is converted from an inactive to an active conformation by photo-isomerization. These methods for modulating Ca and nucleotide agonist concentrations with illumination are intended to open the way for approaches such as relaxation analysis that are not available to us with present methods of manipulating these intracellular messengers.